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. 2017 Sep 12;7:11367. doi: 10.1038/s41598-017-11309-7

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Increased mitochondrial ROS contributes to cell death induced by MKK6 expression. U2OS cells expressing a Tet-regulated construct were either mock treated (control) or treated with tetracycline for the indicated times to induce the expression of constitutively active MKK6. (a) Cell survival was assayed at the indicated times using Annexin V/PI staining. Live cells were determined as the cell population Annexin V⁻ and PI⁻. (b) Intracellular ammonia accumulation was measured in control and MKK6 expressing cells at 32 h and is presented as nmol / 5 × 10⁴ cells. (c) Total ROS levels were analysed at the indicated times after MKK6 induction using the DCFH-DA probe. Values are presented as fold change of mean fluorescence of DCF versus the control cells. (d) ROS levels in control and MKK6 expressing cells treated for 24 h with the p38α inhibitors PH797804 (PH) or SB203580 (SB) were analysed using the DCFH-DA probe. Values are presented as fold change in the mean fluorescence of DCF. (e) Mitochondrial ROS levels in control and MKK6 expressing cells either mock-treated or treated with SB were evaluated at 24 h using the MitoSox probe. Values are represented as fold change in the mean fluoresce of MitoSox normalized towards control cells.